High concentration white blood cells as a therapeutic product

ABSTRACT

A therapeutic product formed from a high concentration of white blood cells having a high degree of cell viability. The white blood cells are sequestered from their normal population presence in whole blood by placing the blood into a container and preventing coagulation of the blood, separating the blood into two components, one of which is extremely rich in white blood cells through the use of a reagent and centrifugation, sequestering the white cell concentration, and freezing the white cells.

This application is a continuation of U.S. application Ser. No.09/313,816, filed May 18, 1999, now U.S. Pat. No. 6,491,678 B1, issuedDec. 10, 2002, which in turn is a continuation of U.S. application Ser.No. 09/128,208, filed Aug. 3, 1998, now U.S. Pat. No. 5,928,214, issuedJul. 27, 1999, which in turn is a continuation of U.S. application Ser.No. 08/349,747, filed Dec. 5, 1994, now U.S. Pat. No. 5,789,147, issuedAug. 4, 1998, the contents of which are incorporated herein byreference.

FIELD OF THE INVENTION

The following invention is directed generally to the therapeuticutilization of white blood cells, a technique for sequestering the whiteblood cells by causing them to coalesce in a population density greaterthan they normally occur in nature, and a method for causing an enrichedconcentration in conjunction with an array of bags oriented in a setthat facilitates both the concentration process and a method forpreserving the white blood cells.

BACKGROUND OF THE INVENTION

It is now recognized that placenta/umbilical cord blood (PB) containslarge numbers of hematopoietic stem and progenitor cells that endow PBwith extraordinary therapeutic capabilities in the reconstitution ofbone marrow damaged as a result of inherited diseases, accidents ormedical procedures. As in the case of ordinary collection of bone marrowfor transplantation, PB contains immune cells potentially capable ofmounting specific responses against the recipients of such transplants,but in contrast to adult immunological cells, those in PB display alower, perhaps much lower tendency to produce damaging immune responsesagainst the recipient. The clinical syndrome produced by the immunoresponses of the graft against the recipient's cells and tissues isdesignated “Graft versus Host Disease” (GVHD). In the typical clinicalsituation, the recipient's own immune response against the graft isabrogated by drugs and irradiation treatments designed to reduce oreliminate the immunological and other hematopoietic cells and thus avoidthe host versus graft immune reaction that would cause rejection of thegraft. It has been proven that the principal targets of these Graftversus Host and Host versus Graft immune reactions are antigens encodedby the genes of the HLA (Human Leukocyte Antigen) system and thatsuccessful outcomes of bone marrow transplants are dependent on thesharing of HLA antigens by donor and recipient. Sibling donors who haveinherited the same paternal and maternal HLA genes present in therecipient are HLA-identical and thus, optimal from this viewpoint.Patients lacking such HLA-identical sibling donors must receivetransplants from more distant relatives or from unrelated donors.Because the HLA system includes several discrete genes each of whichdisplays an extremely large number of antigenically different variantsin the population, such distant relative-donor or unrelated-donortransplants must be expected to contain a variable number of HLAincompatibilities unless they are selected from among potential donorsby identifying the specific variants present in each and choosing donorswhose HLA antigens match those of the recipient. To perform thisselection with significant probability of success, it is necessary tohave access to large panels of potential donors whose HLA antigens areknown. In the case of unrelated donor PB, this requires establishing abank of frozen HLA-typed units collected from random placentas.Heretofore, the most widely accepted method for freezing PB consisted ofadding to the whole PB unit an equal volume of a cryopreservativesolution, with the double disadvantage that the volume of eachcryopreserved unit becomes very large and that a relatively large amountof possibly deleterious cryopreservative is eventually administered tothe recipients of such PB units. Administration of cryoprotectant andhemoglobin from erythrocytes destroyed by using a freezing and thawingmethod designed to protect the stem and progenitor cells but not theerythrocytes may have toxic effects generally and especially on specificorgans such as the kidney of the recipient. In addition, there is thelogistical consequence that a large number of freezers would be neededto contain useful numbers of the large volume frozen units in reserve,with the attending increase in up-front and running costs. Theapplicants have developed a practical method that allows a substantialreduction of the volume of PB Units by eliminating the unneeded maturered blood cells and an equivalent volume of plasma. This submissiondescribes this method and a specially designed set of plastic bags andconnecting tubes intended to facilitate the accomplishment of thedesired concentration of the needed stem cells and progenitor cells withminimal manipulation and risk of contamination. Essentially, this methodwill allow an experimental, time consuming laboratory process to becomea routine procedure in blood banks.

The following submission reflects the state of the art of whichapplicant is aware insofar as these documents appear germane to thepatent process. However, it is respectfully stipulated that none ofthese patents teach singly nor render obvious when considered in anyconceivable combination the nexus of the instant invention as set forthhereinafter.

INVENTOR U.S. Pat. No. ISSUE DATE Tenczar, Jr. 3,187,750 06/1965Williams 4,332,122 06/1982 Pattillo, et al. 4,937,194 06/1990 Boyse, etal. 5,004,681 04/1991 Carmen, et al. 5,104,788 04/1992 Bauman, et al.5,154,716 10/1992 Boyse, et al. 5,192,553 03/1993

OTHER PRIOR ART (Including Author, Title, Date, Pertinent Pages Etc.)Pablo Rubinstein, Richard E. Rosenfield, John W. Adamson and Cladd E.Stevens (The Lindsley F. Kimball Research Institute of The New YorkBlood Center); Stored Placental Blood for Unrelated Bone MarrowReconstitution; May, 1993; entire paper.

SUMMARY OF THE INVENTION

The therapeutic product of the present invention is advantageous, first,because it recovers all or almost all of the stem and progenitor cellsof the original collection of PB in a small and uniform volume thatrequires minimal and predictable storage space, second, because itpermits a consistent methodology for processing PB units which resultsin a routinely dependable product with less dependence on operator skilland third, because the potentially deleterious effects of thecryoprotectant and of the free hemoglobin are minimized.

One first aspect to the nature of the product improved according to thepresent invention involves the methodology by which the white bloodcells (which include the hematopoietic stem and progenitor cells) areseparated from the bulk of other components in the whole PB and themanner in which the viability of such, white cells is preserved byavoiding exposure to bacterial and fungal contamination, potentiallydamaging chemical agents, excessive centrifugal forces and osmoticimbalances. Typically, bacterial and/or fungal contamination occurs whenPB or white blood cell suspensions derived from PB are exposed toambient air in the course of preparatory manipulations; chemical damageis possible when certain chemicals are used to lyse the accompanying redblood cells or to aggregate white cells; and physical damage follows theuse of excessive centrifugal speed in separation of the cellularcomponents of the blood according to their density, by centrifugalstratification. In addition, the method according to the presentinvention provides for avoidance of prolonged exposure of the separatedwhite blood cells to cryopreservation solutions at room temperature, anexposure that results in decreased viability of the white blood cellsand of the stem and progenitor cells contained therein because ofosmotic imbalances and, possibly, other toxic effects of theintracellular cryoprotectants themselves.

Another aspect of the present invention involves the set ofinterconnected plastic containers (designated as bags). The set underthe present invention permits a selective concentration of the whiteblood cells and of the stem and progenitor cells contained thereinwithout reducing their normally high viability and freedom fromcontamination by infectious organisms from the environment. Whole PB iscollected into a mother bag and is subsequently processed through aseries of bags of appropriate chemical structure and physical shape andcapacity culminating in storage of a separated fraction containing mostof the white blood cells of the collected PB in liquid nitrogen at −196C. inside a specially constructed freezer bag. Intervening steps includethe addition of substances that enhance the aggregability of red bloodcells and the separation of components by transferring supernatants intoconnected satellite bags. A special bag and its connecting assemblypermits the addition of measured amounts of cryoprotectant to theseparated white blood cell concentrate. This connecting assembly allowsthe cryoprotectant to be added to the white cells at a precise, slowspeed required to maintain optimal cell viability.

The bag which is to be used for freezing and storage includes aplurality of connected, but detachable compartments for sequestration ofthe white blood cells into different discrete chambers. One chamber, themain compartment, is intended to keep the bulk of the white blood cells.A smaller compartment lends itself to the storage of a smaller fractionof the bag contents which may be separated from the main compartmentwithout thawing, and extemporaneously detached from it for separatethawing and subsequent in vitro expansion of the hematopoietic stem andprogenitor cell populations contained in the corresponding fraction ofthe white blood cells. A third and subsequent chamber contains verysmall aliquots of the white blood cell suspension and are intended toserve as detachable samples for testing the aptness of the unit to betransplanted or assessing its suitability as donor tissue for a specificrecipient. The freezing bag also includes indicia on the outer surfaceof each of its detachable areas for identification of the specific unitthat will be stored in it, to facilitate storage and retrieval fromdesignated sectors of cryogenic storage depots. Means are also providedin an exterior surface of the freezer bag to facilitate the placing andremoving of the freezer bag into and from, respectively, its assignedstorage location by automated instrumentation.

OBJECTS OF THE INVENTION

Accordingly, it is a primary object of the present invention to providethe means for preparing PB derived hematopoietic stem and progenitorcells in a novel and therapeutically more useful form. The productbecomes a bag containing a high concentration of white blood cellshaving a high degree of cell viability.

A further object of the present invention is to provide a novel anduseful method for generating the therapeutic product according to theprevious object.

A further object of the present invention is to provide an aseptic andinterconnected bag set for use in conjunction with the method ofdeveloping the therapeutic product hereinabove.

A further object of the present invention is to provide a freezerstorage bag configured to contain the therapeutic dose in acryoprotected environment for protracted periods of time until neededfor dosage.

A further object of the present invention is to provide a freezer bag asnoted above provided with a plurality of compartments in which thetherapeutic dose has been sequestered so that various aliquots can bestrategically excised from the freezer bag for several purposes.

Viewed from a first vantage point, it is an object of the presentinvention to provide a system for developing placental stem cells,comprising in combination: a first blood bag adapted to receive bloodfrom a placenta therewithin, means within the blood bag to preventcoagulation, reagent means removably coupled to the blood bag, means forseparating supernatant from the first blood bag and into a white cellbag, means for separating white cells from plasma in the white cell bag,a plasma bag removably coupled to the white cell bag for receiving theexpressed plasma from the white cell bag, cryoprotectant meansoperatively coupled to the white cell bag, and a stem cell freezing bagoperatively coupled to the white cell bag for transferring contents fromthe white cell bag to the stem cell freezing bag.

Viewed from a second vantage point, it is an object of the presentinvention to provide a method for preparing concentrated and partiallypurified white blood cell suspensions containing placental stem cells,comprising the steps of: placing blood from a placenta into a firstblood bag, preventing coagulation within the blood bag, coupling reagentmeans into the blood bag, centrifuging and separating white blood cellrich supernatant from the first blood bag and placing the supernatantinto a white cell bag, separating white cells from plasma in the whitecell bag, removably coupling a plasma bag to the white cell bag andexpressing the plasma from the white cell bag into the plasma bag.Coupling cryoprotectant means to the white cell bag, transferringcontents from the white cell bag to a stem cell freezing bag, andfreezing the stem cell freezing bag with its contents follows.

Viewed from a third vantage point, it is an object of the presentinvention to provide a therapeutic product comprising at least 80% ofthe white blood cells (including stem and progenitor cells) withviability greater than 90% and fewer than 10% of the red blood cells inthe original PB collection.

These and other objects will be made manifest when considering thefollowing detailed specification when taken in conjunction with theappended drawing figures.

BRIEF DESCRIPTION OF THE DRAWING FIGURES

FIG. 1 is a schematic view of the stem cell processing bag set accordingto the present invention.

FIG. 2 is a detailed view of the freezing bag shown in FIG. 1.

FIG. 3 is a view similar to FIG. 2 showing the interior of the freezingbag.

FIG. 4 is a flow chart the method according to the present invention.

DESCRIPTION OF PREFERRED EMBODIMENTS

Referring to the drawings now, wherein like reference numerals refer tolike parts throughout various figures, reference numeral 100 is directedto an apparatus according to the present invention.

In essence, the apparatus 100 may be viewed as three arrays of bagscollectively defining a bag set. Individual bags are provided withremovable connection means to assure selected admission into the severalbags only under aseptic conditions. In a preferred form of theinvention, the array of bags 100 includes six bags: a blood bag 10defining a first array; a reagent bag 20, a white cell bag 30, a plasmabag 40 and a cryoprotectant bag 50 defining a second array; and a stemcell freezing bag 60 defining a third array. Cord blood (i.e. blood fromthe placenta and umbilical cord) is admitted to the blood bag 10 whichhad previously been dosed with an anticoagulant. Next, the second arrayis connected to the blood bag 10. A separation reagent is admitted tothe blood bag via conduit 26 from reagent bag 20. Centrifuging blood bag10 follows. Supernatant containing the white blood cells is expressedoff into the white cell bag 30, whereupon further centrifugation takesplace. Next, supernatant plasma is expressed off into the plasma bag 40leaving sedimented white blood cells in white cell bag 30.Cryoprotectant from the cryoprotectant bag 50 is transferred via conduit59 to the white cell bag 30 slowly. Subsequently, the contents of thewhite cell bag 30 are transferred to the stem cell freezing bag 60 whichis thereafter frozen and stored in liquid nitrogen for subsequent use.

More specifically, and with reference to FIG. 1, whole, placental, andumbilical cord blood is collected into a blood bag 10 provided with ananticoagulant such as Citrate, Phosphate and Dextrose (CPD). Assume, forthe sake of explanation, that one hundred (100) milliliters of blood areplaced within the blood bag. Typically, cord blood will exhibit a ratioof one thousand (1,000) red cells to each “non-red” cell (forsimplicity, assume the non-red blood cell can be labeled white bloodcells). Naturally, the main recognizable and functionally capable cellscirculating in blood include erythrocytes, neutrophilic, eosinophilic,and basophilic granulocytes; B- T- and non B- non T-lymphocytes;monocytes and platelets. These mature cells derive from and arereplaced, on demand, by morphologically recognizable dividing precursorcells for the respective lineages such as erythroblasts for theerythrocyte series, myeloblasts, promyelocytes and myelocytes for thegranulocyte series, and megakaryocytes for the platelets. The precursorcells derive from more primitive cells that can simplistically bedivided into two major subgroups: stem cells and progenitor cells. Ofcourse, neonatal blood has other cellular constituents which will not bediscussed here so as not to obscure the essence of the invention. Theblood bag 10 includes at least two access portals. A first portal 2receives the cord blood whereupon the access portal 2 is sealed.Typically sealing includes a heat seal to insure asepsis. A secondportal 4 is provided which communicates with a spike 22 coupled viaconduit 26 to a separation reagent bag 20 and through conduit 32 to thewhite cell bag 30 from the second array of bags discussed above. Inaddition, the blood bag 10 may also be provided with a third access 6which may include a sample tube, should it be found desirable to placeinto storage an exemplar of the cord blood which was originally drawn.Access 6 may also provide alternative connections to bag 10.

Once the cord blood has been admitted into the blood bag 10, theadmixture with an anticoagulant such as CPD prevents the clotting of theplacental blood and readies the blood for admixture with a reagentcontained within reagent bag 20. After the admission of the reagent tothe blood bag and thorough mixing, the bag is centrifuged at a precisespeed and the white-cell-rich supernatant is expressed into the whitecell bag 30. The reagent is intended to facilitate the sedimentation ofthe red blood cells which is greatly accelerated by a very lightcentrifugation step (50×G×5 min.). The effects of the addition ofseparation reagent and centrifugation are to produce a supernatant whichcontains eighty to ninety-five percent (80–95%) of the white blood cellsand less than ten percent (10%) of the red blood cells of the originallycollected blood. This reduces the presence of red cells (compared towhite cells) by approximately ninety percent (90%). In the white cellbag, the red cell to white cell count ratio is now reduced toapproximately one hundred (100) to one (1).

Typically, reagents which promote effective separation of the red bloodcells from the white blood cells operate on the basis of mechanismswhich can be the subject matter of some speculation as to the physicalprocess or model that describes the separation process. One vantagepoint advances the premise that the addition of the reagent raises thedielectric strength of the suspension medium and then, itscharge-dissipating capacity, so that the tendency for the red bloodcells to remain in uniform suspension is disturbed. Another view is thatthe polymeric molecule of the reagent binds to two or more red bloodcells, causing them to aggregate and form characteristic “rouleau” i.e.,loose clumps of red blood cells stacked together by the flat aspects oftheir discoidal surface. The effect, however, irrespective of thephysical model that one envisions, is that separation between the redand white cells is possible with relatively minor, gentle and briefcentrifugation. This accelerates the settling of the red cells andpreserves the white cells in the suspended, unmodified state. In apreferred embodiment, once the reagent from bag 20 has been placedwithin the blood bag 10, centrifugation at fifty (50) gs forapproximately five (5) minutes provides effective separation.

Reagents which change the charge dissipation characteristic or alter thedielectric strength of the constituent components can be selected from arelatively broad range of suitable substances. A six percent (6%)concentration of Heptastarch is presently preferred both due toefficacy, cost, and wide spread utilization in clinical bloodprocessing. However, similar natural polymers such as dextrans,gelatins, modified or unmodified starches or synthetics such aspolyethylene glycol or polyvinyl-pirrolydone and many others couldconceivably be substituted as conditions warrant. A similar effect mayalso be obtained with substances whose molecules attach with highavidity to two or more red cells such as antibodies and lectins. In anyevent, any one of these red cell-cryoprecipitating reagents contained inthe reagent bag is dispensed from the reagent bag 20 via outlet 24through branch passageway 26 and through the outlet spike 22 received byportal 4 or, alternatively, portal 6, into the blood bag 10.

Mixing of the reagent with the blood in the bag 10 followed by gentlecentrifugation results in a separation in which the supernatant composedof plasma, most of the white blood cells and a small fraction of the redblood cells, is expressed off into the white cell bag 30 via a branchpassageway 32 communicating between the spike 22 and the bag 30 with aT-adapter 34 which allows a bifurcation between the branch 26 and thebranch 32. The bulk of the red blood cells remain in bag 10. Theenriched white cell mixture is prevented from entering the reagent bagby means of a clamp 28 operatively engaged on the branch passage 26. Theenriched white cell mixture in the white cell bag 30 at inlet 36 is nowready for further processing.

As an example, assume one hundred (100) milliliters of PB had beenoriginally collected into blood bag 10. A preferred embodiment providesa reagent bag 20 with a sufficient volume of Hydroxyethyl starch(Heptastarch, Dupont) to provide for the addition of a volume equal toone-fifth (⅕) that of the PB collection into bag 10. In this example,one-fifth (⅕) of one hundred (100) milliliters equals twenty (20)milliliters. Typically, seventy (70) milliliters of white cell enrichedsupernatant plasma (containing the reagent solution) will be producedwhich will be expressed into the white cell bag 30. Once there, thecontents are subjected to further centrifugation at four hundred(400)×G×ten (10) minutes. Typically, of the seventy (70) millilitersthat had been admitted into the white cell bag 30, fifty-five (55)milliliters will be expressed off thereafter into a plasma bag 40,leaving approximately fifteen (15) milliliters of highly-enriched whitecell product in bag 30. The supernatant transferred to bag 40 containsthe bulk of the plasma, anticoagulant and reagent and essentially nocells.

The white cell bag 30 includes an outlet portal 38 that communicateswith the plasma bag 40 via a branch conduit 42 having a T-adapter 44 anda constrictor 46 in line. The supernatant is expressed from white cellbag 30 via conduit 42 to the plasma bag 40 via its own portal 48. Oncethe supernatant has been received into the plasma bag 40 it is sealedoff and the plasma bag 40 is disconnected from the white cell bag 30.

Cryoprotectant from cryoprotectant bag 50 is next admitted into thewhite cell bag 30. Cryoprotectant bag 50 includes an outlet 52, a branchpassageway 54 and a constrictor element 56 on the line 54 in fluidiccommunication with the portal 38 of the white cell bag 30 throughT-adapter 44. Typically, three point eight (3.8) milliliters ofcryoprotectant is admitted into the fifteen (15) milliliters containedwithin the white cell bag 30. It is extremely desirable to admit thecryoprotectant into the white cell bag 30 at a relatively slow rate.Typically, the three point eight (3.8) milliliters of cryoprotectant isadmitted into the bag over a twenty (20) minute interval, whilecontinuously mixing the cryoprotectant with the contents of the whitecell bag by hand or with an orbital shaker. A preferred cryoprotectantsolution includes Dimethyl Sulfoxide DMSO (an intracellularcryoprotectant) diluted to fifty percent (50%) with dextran anextracellular cryoprotectant. One feature of the instant invention isthat the constrictor element 56 determines that the intracellularcryoprotectant can only enter white cell bag 30 very slowly. Thus, theintracellular cryoprotectant increases its concentration and permeatesthe white cell mixture contained within the white cell bag 30 withoutcausing damage to the cells. In order to effect same, a meteringinstrumentality 58 may be interposed in the branch 54 instead of theconstrictor element 56 (should the constriction not provide a constantflow rate) and in fluid communication with the portal 38. The meteringinstrumentality 58 can be a pump. Alternatively the cryoprotectant bagand pump arrangement can be replaced with a syringe or other meteringapparatus which facilitates the slow addition of cryoprotectant to thewhite cell bag 30.

The physical analogy for the cryoprotectant is that the DMSO penetratesthrough the white cell membrane and reduces the capacity ofintracellular water as it freezes to crystallize intracellularly andinflict damage to the cell walls. Dextran and other extracellularcryoprotectants such as diverse kinds of soluble starches, proteins andsugars are believed to provide extracellular layers around white cellsthat insulate the cells from the tendency of the water to form crystalsduring the freezing process and to develop excessive extracellularhyperosmolarity, both of which might reduce cell wall integrity andcellular viability. By providing the cryoprotectant at a measured rate,over a relatively long period of time, cell viability will have beenmaximized by providing ample time for the DMSO to diffuse into cells andto reach equilibrium across the cell membrane and for the dextran to behomogeneously diluted in the surrounding plasma.

As an example of the preferred embodiment, three and eight/tenth (3 and8/10) milliliters of cryoprotectant is added to the fifteen (15)milliliters of white cells in the white cell bag 30. This additionbrings the concentration of DMSO to ten percent (10%) in bag 30. Whitecell bag 30 has another outlet 62 which receives a spike 64 from thestem cell freezing bag 60 in an aseptic manner. The white cell bag 30communicates with stem cell freezing bag 60 via conduit 66 controlled byclamp 67. The cryoprotected white cell mixture is received into the stemcell freezing bag 60 via portal 68. The portal 68 is speciallyconfigured to include a stand tube which allows a standing column ofstem cell mixture to be retained therewithin for sequestering into aseries of compartments 70, each spaced from the other by heat seals 72.These specimens 70 can be used for pre-infusion confirmation of anoptimum HLA match or other tests, once a particular stem cell freezingbag 60 has been chosen as appropriate for the putative recipient.

The stem cell freezing bag 60 is further characterized by having aplurality of compartments within the main body of the bag 60, eachcompartment provided with indicia 75 thereon for identification of thespecific unit, establishing a form of chain of custody. Moreparticularly, the stem cell freezing bag 60 includes at least a firstmajor portion 74 and a second minor portion 76. Typically, the ratiobetween the major portion 74 and the minor portion 76 is eighty percent(80%) major portion and twenty percent (20%) minor portion. Theseportions of bag 60 are delimited by heat seal 80 and, after filling,contribute to dividing the stem cell freezing bag into two, intimatelyattached, but independent white cell containers once heat seals at bothlocations 82 are executed.

Each portion is in communication with its own outlet. The major portion74 is in communication with its portal 84 while the minor portion 76communicates with its own portal 86. In addition, the heat seal locationmay include a line of demarcation 81 defining a scoreline which allowsthe major portion 74 to be severed, without thawing, from the minorportion 76. It is contemplated that the stem cells contained in theminor portion 76 can be allocated for other uses, such as for increasingthe numbers of useful cells by culturing the stem and progenitor cellsin a propagation medium. The stem cells in major portion 74 are leftundisturbed for administration as transplants. The freezer bag 60 andstand tube/portal 68 have negligible thickness. The purpose of thisparticular geometry is to assure that the white cells in compartments76, 74 and 70 all maintain a uniform and narrow thickness so thatsubsequent freezing regimens achieve near identical controlled ratefreezing conditions.

In a preferred embodiment, approximately nineteen (19) milliliters oftherapeutic product are contained within the freezing bag 60. The stemcell freezing bag 60 is gradually frozen to an extremely low temperaturesuch as in liquid nitrogen for permanent storage. This preserves thestem cells in a state such that, upon thawing, they are recovered inquantity and exhibit a high degree of cell viability.

Once it has been determined that the given stem cells within a freezingbag 60 are to be used in a transplant procedure, the stem cells arefirst thawed to a temperature where the stem cells and constituentcomponents change phase back from a solid to a liquid. Next, the stemcells are washed to remove the cryoprotectant which was added prior tofreezing. Preferably, the wash is intended to remove the DMSO by usingan isotonic fluid, preferably a colloid. For example, a mixture havingfive percent (5%) albumin and ten percent (10%) dextran in a salinesolution is used to dilute the DMSO in the extracellular environment andsecondarily reduce its concentration inside the white blood cells.Subsequently, the mixture is centrifuged at four hundred (400) gs forten (10) minutes with the supernatant expressed therefrom.

As mentioned supra, the enriched white cells were present in volume atapproximately fifteen (15) milliliters prior to the addition of threepoint eight (3.8) milliliters of cryoprotectant. When placed in the stemcell freezing bag, about four (4) milliliters were placed in thesecondary compartment 76 and fifteen (15) milliliters were retained inthe primary container 74. In actuality, somewhat less than the four (4)milliliters are allocated as is just described because the stem cellsamples contained within compartments 70 may contain collectively up toone (1) milliliter. In any event, the thawed white blood and stem cellsuspension prior to washing contained ten (10%) cryoprotectant byvolume. After the dilution, spinning and expressing off the supernatantthe sedimented stem cells (typically in a volume less than three (3)milliliters) are diluted once again to a volume adequate foradministration to the recipient, fifteen (15) milliliters or more. Thissecond dilution reduces the concentration of DMSO to below one percent(1%). Therefore, the quantity of DMSO retained is in the order toone-tenth ( 1/10) gram. This is very much less, compared with the priorart which typically may have involved two hundred (200) milliliters often percent (10%) DMSO i.e. twenty (20) grams of this compound.

In addition, the therapeutic dose described in the disclosed inventionhereinabove has a special efficacy because the processing describedhereinabove has removed from the whole blood, the bulk of the red cells,plasma, cryoprotectant, free hemoglobin, etc. which heretofore haveexhibited adverse consequences on the recipient and has restored theosmolarity of the stem and progenitor cells to the normal range of threehundred (300) milliosmols from the over one thousand (1000) milliosmolsof ten percent (10%) DMSO solution.

It is to be noted that the stem cells that are stored in freezing bagsmust be kept at extremely low temperatures such as those achievableusing liquid nitrogen. By providing white stem cells in twenty (20)milliliter quantities, the problems that would have existed before inthe provision of storage space for units with ten fold larger volumes ofcryopreserved placental blood (whole) will have been solved by thesmaller storage requirement of separated white blood cells associatedwith the instant invention.

One attribute of the instant invention is that the therapeutic doseinvolves a relatively low level of DMSO in the finished product that isto be administered. A second attribute involves the fact that a ten (10)fold lower concentration of red blood cells are contained in a unitwithout significant loss of stem and progenitor cells. The lower redblood cell numbers reduce the presence of hemoglobin in the thawedspecimen and decrease the problems associated with red blood cellincompatibilities. Further, the viability of the white cells containedin the dose after thawing is typically three (3) to four (4) fold higherthan in the prior art, particularly after administration and dilution inthe recipient's own plasma. Experimentally, thawed white cells arediluted in twenty (20) milliliters of plasma prior to counting forviability. In prior art, unwashed white cell viability was typically ofthe order of twenty percent (20%). According to the present invention,using the DNA fluorescence stain or other viability tests, themononuclear cells are much greater than twenty percent (20%), typicallygreater than ninety percent (90%) viable. When stem and progenitor cellsare cultured in vitro from such white cell concentrates after thawing asdescribed, the number of viable cells estimated by the number ofcolonies formed is also greater than ninety percent (90%) of theoriginal numbers.

While the previous discussion has focused on the desirability of usingcord blood from placental stem cells, other peripheral stem cells canalso be processed in a similar manner to provide benefits. Further,having thus described the invention it should be apparent that numerousstructural modification and adaptations of the bag set, the chemicalnature of the reagents and cryoprotectants and the details of theprocessing steps, may be resorted to without departing from the scopeand fair meaning of the instant invention as set forth hereinabove andas described hereinbelow by the claims.

1. A therapeutic product separated from a sample of cord blood orplacental blood, wherein the therapeutic product consists essentially ofat least 80% of white blood cells contained in the sample of cord bloodor placental blood, less than all of plasma contained in the sample ofcord blood or placental blood, less than 10% of red blood cellscontained in the sample of cord blood or placental blood, and a redblood cell sedimentation reagent that facilitated separation of the redblood cells from the white blood cells contained in the sample of cordblood or placental blood, and wherein the therapeutic product has avolume of 10 milliliters to 20 milliliters.
 2. The therapeutic productof claim 1, wherein the therapeutic product further consists of acryoprotectant.
 3. The therapeutic product of claim 2, wherein thecryoprotectant comprises dimethyl sulfoxide.
 4. The therapeutic productof claim 2, wherein the cryoprotectant comprises dextran.
 5. Thetherapeutic product of claim 1, wherein the therapeutic product furtherconsists of an anticoagulant.
 6. The therapeutic product of claim 5,wherein the anticoagulant comprises Citrate, Phosphate, and Dextrose(CPD).
 7. The therapeutic product of claim 1, wherein the therapeuticproduct has an osmolarity of not more than 300 milliosmols.
 8. Thetherapeutic product of claim 1, wherein the red cell to white cell countis approximately one hundred (100) to one (1).
 9. A therapeutic productseparated from a sample of cord blood or placental blood, wherein thetherapeutic product comprises at least 80% of white blood cellscontained in the sample of cord blood or placental blood, less than allof plasma contained in the sample of cord blood or placental blood, lessthan 10% of red blood cells contained in the sample of cord blood orplacental blood, a red blood cell sedimentation reagent that facilitatedseparation of the red blood cells from the white blood cells containedin the sample of cord blood or placental blood, and dimethyl sulfoxidediluted to 50% with dextran.
 10. A therapeutic product separated from asample of cord blood or placental blood, wherein the therapeutic productcomprises at least 80% of white blood cells contained in the sample ofcord blood or placental blood, less than all of plasma contained in thesample of cord blood or placental blood, and a red blood cellsedimentation reagent that facilitated separation of the red blood cellsfrom the white blood cells contained in the sample of cord blood orplacental blood, and wherein the red cell to white cell count isapproximately one hundred (100) to one (1).